Oxidative pathways of apo, partially, and fully Zn(<scp>II</scp>)‐ and Cd(<scp>II</scp>)‐metalated human metallothionein‐3 are dominated by disulfide bond formation

Oxidative stress is a key component of many diseases, including neurodegenerative diseases such as Alzheimer's disease. Reactive oxygen species (ROS) such as hydrogen peroxide and nitric oxide lead to disease progression by binding to proteins and causing their dysregulation. Metallothionein-3 (MT3), a cysteine-rich brain-located metalloprotein, has been proposed to be a key player in controlling oxidative stress in the central nervous system. We report data from a combination of electrospray ionization mass spectrometry (ESI-MS), ultraviolet (UV)-visible absorption spectroscopy, and circular dichroism spectroscopy that identify the oxidation pathway of MT3 fully bound to endogenous Zn(II) or exogenous Cd(II) together with the partially metalated species. We characterize the intermediate species formed during the oxidation of MT3, which is dominated by disulfide bond formation. We report the rates of oxidation. For both fully and partially metalated MT3, MT3 is oxidized at 5 to 10 times the rate of MT1, a similar but kidney-expressed isoform of MT. As oxidation progresses, MT3 follows a domain-specific demetallation pathway when it is fully metalated, and a domain-independent pathway when partially metalated. This suggests the presence of a significant susceptibility toward oxidation when MT3 is partially metalated, and, therefore, a possible protective role of Zn(II) when fully metalated. With the evidence for the rapid oxidation rate, our data support the proposals of MT3 as a key antioxidant in physiology.